The basis of gene therapy is to deliver a functional gene to tissues where the respective gene activity is missing or defective. Among the approaches to accomplishing gene therapy has been the use of recombinant viral vectors which have been genetically engineered to carry a desired transgene. These viral-based vectors have advantageous characteristics, such as the natural ability to infect the target tissue. However, implementation of existing viral vectors are impeded by several limitations as well.
For example, retrovirus-based vectors must integrate into the genome of the target tissue to allow for transgene expression (with the potential to activate resident oncogenes) while vector titers produced in such systems are significantly less than in some other systems. Because of the requirement for integration into the subject genome, the retrovirus vector can only be used to transduce actively dividing tissues. Further, many retroviruses have limited host tissue specificity and cannot be employed to transduce more than a few specific tissues of the subject.
Adenovirus vectors hold great promise for gene therapy. Adenovirus vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney, muscle (skeletal and cardiac), respiratory, and nervous system tissues. See, e.g., Askari et al., Gene Ther. 3:381-388 (1996); Barr et al., Gene Ther. 1:51-58 (1994); Engelhardt et al., Hum. Gene Ther. 4:759-769 (1993). Using transcomplementing packaging cell lines, first generation Adenovirus vectors can be grown and concentrated to high titers (&gt;10.sup.13), which contributes to their ability to transduce large numbers of target cells after in vivo administration. Ragot, et al., Nature 361:647-50 (1993).
First generation adenovirus vectors also have a comparatively large carrying capacity (i.e., up to about 8.0 kb). The ability of first generation Adenovirus vectors to allow expression of transduced genes episomally for extended periods of time in immune-incompetent and sometimes immune-competent animals without the need for integration into the vector genome (Vincent, et al., Nat Genet: 5:130-34 (1993); Tripathy, et al., Nature Medicine 2:545-50 (1996)) allows them to transduce mitotically quiescent cells as well as actively dividing cells. Finally, live Adenovirus preparations have been used for the vaccination of military recruits, and Ad strains 2 and 5 (most commonly used for vector development) are not associated with severe disease.
Despite the advantages discussed above, first generation, E1-deleted adenovirus virus vectors are limited in potential therapeutic use for several reasons. First, due to the size of the E1 deletion and to physical virus packaging constraints, first generation adenovirus vectors are limited to carrying approximately 8.0 kb of transgene genetic material. While this compares favorably with other viral vector systems, it limits the usefulness of the vector where a larger transgene is required. Second, infection of the E1-deleted first generation vector into packaging cell lines leads to the generation of some replication competent adenovirus particles, because only a single recombination event between the E1 sequences resident in the packaging cell line and the adenovirus vector genome can generate a wild-type virus. Therefore, first-generation adenovirus vectors pose a significant threat of contamination of the adenovirus vector stocks with significant quantities of replication competent wild-type virus particles, which may result in toxic side effects if administered to a gene therapy subject.
The most difficult problem with adenovirus vectors is their inability to sustain long-term transgene expression, secondary to hose immune responses that eliminate virally transduced cells in immune-competent animals. Gilgenkrantz et al., Hum. Gene Ther. 6:1265-1274 (1995); Yang et al., J. Virol. 69:2004-2015 (1995); Yang et al., Proc. Natl. Acad. Sci. USA 91:44074411 (1994); Yang et al., J. Immunol. 155: 2565-2570 (1995). While immune responses have been demonstrated against the transgene-encoded protein product (Tripathy et al., Nat. Med. 2; 545-550 (1996)), it has also been demonstrated that adenovirus vector epitopes are major factors in triggering the host immune response. Gilgenkrantz et al., Hum. Gene Ther. 6:1265-1274 (1995); Yang et al., J. Virol. 70: 7209-7212 (1996). It has been repeatedly demonstrated that transgene such as the bacterial .beta.-galactosidase gene are highly immunogenic when transduced by adenovirus vectors, in contrast to other delivery systems (e.g., direct DNA injection or adeno-associated virus administration), where an immune response against the immunogenic transgene is lacking and transgene expression persists. Woff et al., Hum. Mol. Genet. 1:363-369 (1992); Xiao et al., J. Virol. 70:8098-8108 (1996).
In addition, E1.sup.- vectors have also been reported to express the adenovirus early genes, undergo genome replication and express the L1-L5 encoded structural genes when utilized in vivo. E. g., Yang, et al., Immunity 1: 433-442 (1994). Because only a single recombination event is required to produce an entirely replication competent virus from the E-1 deletion, the exaggerated immune response may also be due in some instances to the contaminating presence of wild type adenovirus virus in the vector preparation. Eg., Rich, Hum. Gene. Ther. 4: 461-476 (1993). Either (or both) of these phenomena result in the production and presence of viral proteins in the transduced cells, possibly creating a higher antigenic profile than other gene therapy vector systems. The presence of these adenovirus viral gene products may contribute to the short duration of transgene expression in cells infected by first generation adenovirus vectors by accelerating the detection and elimination of adenovirus vector infected cells by the host immune system.
Accordingly, there remains a need in the art for improved adenovirus vector systems that address the limitations of existing systems.